Use of 5-substituted nucleosides and/or prodrugs thereof in the resistance-free treatment of infectious diseases

ABSTRACT

The present invention relates to the use of 5-substituted nucleosides and/or the prodrugs thereof together with at least one active substance in order to produce a drug or combination preparation for resistance-free therapy of infectious diseases caused by bacteria or protozoa.

[0001] Resistance development relative to treatment with medicines represents a universal defence mechanism of microorganisms, animals and plants. In humans, this defence mechanism is found in tumours. Generally, multiplication (amplification) of specific genes leads to the development of resistance. This gene amplification causes the overproduction of a gene product which directly or indirectly reduces the effect of the drug. Resistance development of Plasmodia (pathogens of malaria), and Leishmania (flagellates as pathogens of cutaneous Leishmaniose i.a.) is based partly on the amplification of the same genes as in human tumours.

[0002] Proof that gene amplification in bacteria leads to resistances has been successful with Proteus bacteria, Escherichia coli, Streptococca and Staphylococca. All are important hospitalism germs and pathogens of urinary tract infections (Romero and Palacios, gene amplification and genome plasticity in procaryotes, Ann. Rev. Genet. 1997). The mechanism underlying gene amplification, recombination, can likewise lead to the development of resistance. This form of resistance development has been proved unequivocally without exception in all types of bacteria but also for human tumours and all organisms which have been examined to date.

[0003] Malaria is a collective term for infections by protozoa of the Plasmodium type. Due to increasing resistance of Plasmodia to chemotherapeutics and of Anopheles mosquitoes to insecticides, the situation is increasingly deteriorating. Both resistances can be caused by gene amplification/recombination.

[0004] 4-aminoquinolines (Chloroquine, Amodiaquine, mepacrine and Sontaquine are used as antimalaria drugs. These are analogues of quinine. Furthermore, there are two groups of antifolates, on the one hand dihydrofolate reductase (DHFR) inhibitors (pyrimethamine and chloroguanide) and on the other hand the sulphones and sulphonamides. Resistances can therefore arise due to amplification of the DHFR gene (Cowman and Lew, 1989; Cowman and Lew, 1990; Tanaka et al., 1990a; Tanaka et al. 1990b; Watanabe and Inselburg, 1994). The amplification of “Multi-drug-resistance” genes is likewise of importance (Foote et al., 1989). The amplification of the pfmdr1 gene is connected for example to resistance to mefloquine, halofantrine and quinine (Wilson et al., 1989; Cowman et al., 1994). As a result of increasing resistance of the Plasmodia to chemotherapeutics and Anopheles mosquitoes to insecticides, the situation is increasingly deteriorating. Both resistances can be caused by gene amplification/recombination. The development of resistance of Plasmodia is intended to be prevented by the prevention of chemotherapy-induced gene amplification.

[0005] Leishmaniases are caused by Leishmania (intracellular parasitic protozoa of the Mastigophora class) and infectious diseases transmitted by Phlebotoma (sand mosquitoes). Resistances to a chemotherapy are based on the amplification of some genes which also play a role in chemoresistance of tumours (Ouellette and Borst, 1991; Grondin et al., 1998; Arana et al., 1998; Heimeur and Ouellette, 1998; Kundig et al., 1999). The development of resistance is intended to be prevented by preventing chemotherapy-induced gene amplification/recombination.

[0006] The use of 5-substituted nucleosides for inhibiting resistance development during cytostatic treatment is already known from DE 195 45 892.

[0007] It is therefore the object of the present invention to make possible a resistance-free treatment of infectious diseases caused by bacteria or protozoa.

[0008] This object is achieved by the generic use with the features of claim 1. The further sub-claims 2 to 20 reveal advantageous developments.

[0009] Resistance-free therapy of infectious diseases from the group of infections caused by bacteria or by protozoa is achieved in that, together with the substance which is active against the infectious disease, 5-substituted nucleosides and/or the prodrugs thereof are administered.

[0010] The active substance and the 5-substituted nucleosides or the prodrugs thereof can thereby occur both in a single formulation and in separate formulations as a combination preparation. A simultaneous, separate or temporally separated application can thereby occur.

[0011] Preferably, the 5-substituted nucleosides are selected from (E)-5-(2-bromovinyl)-2-deoxyuridines (BVDU), the salts thereof, the protective forms thereof and the prodrugs thereof. The compound of formula 1 can thereby be used preferably as prodrug.

[0012] Antibiotics are used in a preferred embodiment as active substances for resistance-free therapy of bacterial infectious diseases. All bacteria indiscriminately are thereby possible as the bacteria triggering infection because of the proved importance of recombination in resistance development and because of the proved importance of gene amplification, e.g. Proteus bacteria, Escherichia coli, Streptococca and Staphylococca.

[0013] For the resistance-free therapy of malaria or of other infections caused by Plasmodia, antibiotics and/or anti-infectives, such as e.g. 4-aminoquinolines, are used as active substances. Chloroquine, Amodiaquine, mepacrine and Sontaquine are used particularly preferably thereby.

[0014] Antifolates are used as further preferred active substance against malaria or other infections caused by Plasmodia. For example, pyrimethamine, chloroguadine, sulphones and sulphonamides are included here.

[0015] For the resistance-free therapy of Leishmaniases, chemotherapeutics, antibiotics and/or anti-infectives are used preferably as active substances. Methotrexate is thereby used as preferred chemotherapeutic.

[0016] The 5-substituted nucleosides or the prodrugs thereof are used preferably in such concentrations that, after administration, a concentration of 5-substituted nucleosides or the prodrugs thereof in the blood of between 0.01 and 10 μg/ml, particularly preferred between 0.05 and 5 μg/ml, results.

[0017] The active substances are administered in standard concentrations according to the type of infectious disease, as are listed for example in current drug lists. Reference is made here in particular to the Red List 2001 (Red List Service GmbH, Frankfurt/Main).

[0018] Administration both of the active substances and also the 5-substituted nucleosides can be effected by injection, orally, rectally, intravaginally, intranasally and/or by local application.

[0019] In addition to the active substance and the 5-substituted nucleosides, there can also be used as further additives, aqueous and non-aqueous solvents, stabilisers, suspension agents, dispersion agents and wetting agents in the formulations. There are possible as additional additives, e.g. polyethylene glycoles, colourants and perfume agents. The formulation can thereby be effected in the form of a fine powder, a powder, a suspension, a solution, an emulsion, a salve or a paste.

[0020] The subject according to the invention is now intended to be explained in more detail with reference to the following examples and FIGS. 1 to 10 without restricting the subject to these examples.

1. EXAMPLE Prevention of Resistance Development in Bacteria

[0021]Escherichia coli J53 was used as Gram-negative model organism in order to test the effectiveness of BVDU. This organism carries the naturally occurring plasmid RP4, on which resistances to Ampicillin, Tetracycline and Kanamycin are encoded.

1.1 Adaptation to Kanamycin

[0022] Kanamycin is a naturally occurring aminoglycoside antibiotic. It is used for the treatment of bacterial infections, if it is not possible to use Penicillin or less toxic antibiotics. Fields of use are inter alia, infections of the bones, the respiratory tracts and of the skin and also complicated urinary tract infections and endocarditis.

[0023] In growth tests with Kanamycin (64 μg/ml), a slow adaptation, i.e. resistance development, of E. coli J53 (RP4) to this concentration of the antibiotic was achieved. In the presence of BVDU (1 μg/ml), an inhibition of this resistance development/adaptation occurred. The results are illustrated in FIGS. 1 and 2.

[0024] The gradual adaptation to 64 μg/ml Kanamycin led to a stable change in the resistance spectrum relative to the antibiotic which was used.

[0025] In order to determine the minimal inhibitory concentrations (MIC), precultures were used initially for which antibiotic-free culture medium had been inoculated directly from frozen glycerine cultures. The strain, which had become resistant to 64 μg/ml Kanamycin had with 128 μg/ml a four times increased MIC relative to the starter strain (FIG. 3). The strain grown in the presence of 1 μg/ml BVDU, which had achieved no resistance to 64 μg/ml Kanamycin, showed growth inhibition already from 16 μg/ml Kanamycin. However even higher antibiotic concentrations still led to the growth of the inoculum. However, the growth inhibition prevented a larger cell density in the bacteria culture being reached.

[0026] In order to test the stability of the specific resistance features of the E. coli strains, the MIC determination was repeated. The already tested precultures were again absorbed for this purpose in antibiotic-free culture medium and tested for their Kanamycin resistance. The resistance spectra of the starter strain and of the strain adapted to 64 μg/ml remained unchanged. However, differences were revealed in the strain which was pretreated with 64 μg/ml Kanamycin +1 μg/ml BVDU. The growth of the inoculum was hereby completely prevented from a concentration of 32 μg/ml Kanamycin. The MIC of this strain corresponded hence to that of the starter strain. The results are illustrated in FIG. 4.

[0027] This means in summary that BVDU prevents the development of resistance to antibiotics and increases the sensitivity to antibiotics in a specific concentration range.

1.2 Adaptation to Amikacin

[0028] Amikacin acts against pathogens which are resistant to the remaining aminoglycosides. It is given in the case of severe infectious diseases of the kidneys, urinary and sexual organs and in infections of the respiratory tracts and of the gastro-intestinal tract.

[0029] In growth tests with Amikacin (0.25 μg/ml), a slow adaptation, i.e. resistance development, of E coli J53 (RP4) to this concentration of the antibiotic was achieved. In the presence of BVDU (2 μg/ml), inhibition of this adaptation occurred (FIG. 5).

[0030] The gradual adaptation to 0.25 μg/ml Amikacin led to a stable change in the resistance spectrum relative to the antibiotic which was used.

[0031] The strain which had become resistant to 0.25 μg/ml Amikacin had with 2 μg/ml an eight times increased minimal inhibitory concentration (MIC) relative to the starter strain. The strain grown in the presence of 2 μg/ml BVDU which had achieved no resistance to 0.25 μg/ml Amikacin, showed growth inhibition already from 0.125 μg/ml Amikacin. This inhibition also prevented a larger cell density in the bacteria culture being reached with higher antibiotic concentrations. The results can be deduced from FIG. 6.

2. EXAMPLE BVDU Conditioned Growth Inhibition After Aminoglycoside/BVDU Pretreatment

[0032] In order to determine the minimal inhibitory concentrations (MIC) of Kanamycin or Amikacin relative to aminoglycoside/BVDU-pretreated E coli J53 (RP4) strains, standard precultures were used which contained no additives in the culture medium (recovery phase). In addition, it was tested in comparative experiments to what extent the addition of BVDU to the antibiotic-free precultures changes the resistant spectrum of the strains.

[0033] The presence of BVDU (1 or 2 μg/ml) in the preculture or/and the small BVDU quantity (0.04-0.05 μg/ml) present due to transfer into the MIC batches sufficed for sustained restriction of the growth of aminoglycoside/BVDU-pretreated E. coli strains even in the absence of the antibiotic. In comparison therewith, the growth without antibiotic in the additive-free preculture corresponded to that of the untreated E. coli strain. The results are illustrated in FIGS. 7 and 8.

[0034] This implies in summary that BVDU also acts alone after removing an antibiotic. BVDU without pretreatment (antibiotic+BVDU) results in no effect and is not toxic.

3. EXAMPLE Prevention of Resistance Development in Protozoa

[0035] Prevention of resistance development in Zooflagellates (Leishmania) by the simultaneous administration of the anti-recombinogenic 5-substituted nucleoside (E)-5-(2-bromovinyl)2′-deoxyuridines (BVDU) with methotrexate.

[0036] Protozoa of the Zooflagellata strain, such as e.g. Trypanosoma as pathogens of sleeping sickness and Leishmania (intracellular parasitic protozoa of the Mastigophora class) as pathogens of Leishmniases are infectious. Resistances to chemotherapy are based both in Trypanosoma (Wilson et al., 1991) and Leishmania (Ouellette and Borst, 1991; Grondin et al., 1998; Arana et al., 1998; Haimeur and Ouellette, 1998; Kundig et al., 1999) on the amplification of some genes which also play a role in the chemoresistance of tumours. The resistance development is intended to be prevented via the prevention of chemotherapy-induced recombination/gene amplification.

[0037] Methotrexate (MTX) resistant cells of Leidshmania donovanii were produced by gradual increase in the MTX concentrations of 5 to 10, of 10 to 50 and of 50 to 100 μm MTX. 5×10⁶ cells/ml respectively were seeded in the growth medium. The cells were diluted again to 5×10⁶ cells/ml for each new batch. The cells were then constantly subjected to the next higher MTX concentration when the cell division rate of the MTX-subjected cells had stabilised to the control level. This was possible after respectively approximately three to four passages. If 1 μg/ml BVDU was added simultaneously to the cultures, then the cell division rate never reached the control level, i.e. the cells developed, in contrast to the cells treated with MIX alone, no resistance to the treatment. The result of this test can be seen in a simplified version in FIGS. 9 and 10. 

1. Use of 5-substituted nucleosides and/or the prodrugs thereof together with at least one active substance in order to produce a drug or combination preparation for resistance-free therapy of infectious diseases caused by bacteria or protozoa.
 2. Use according to claim 1, characterised in that 5-substituted nucleosides or prodrugs thereof and the active substance occur in a single formulation.
 3. Use according to claim 1, characterised in that 5-substituted nucleosides or prodrugs thereof and the active substance occur in separate formulations.
 4. Use according to one of the claims 1 to 3, characterised in that the 5-substituted nucleosides are selected from (E)-5-(2-bromovinyl)-2-deoxyuridines (BVDU), the salts thereof, the protective forms thereof and the prodrugs thereof.
 5. Use according to claim 4, characterised in that the compound of Formula 1 is used as prodrug.


6. Use according to one of the claims 1 to 5, characterised in that antibiotics are used as active substances for resistance-free therapy of bacterial infectious diseases.
 7. Use according to claim 6, characterised in that the bacteria are selected from the group of Proteus bacteria, Escherichia coli, Streptococca and Staphylococca.
 8. Use according to one of the claims 1 to 5, characterised in that antibiotics and/or anti-infectives, such as e.g. 4-aminoquinolines, are used as active substances for resistance-free therapy of malaria or other infections caused by Plasmodia.
 9. Use according to claim 8, characterised in that the quinine derivatives are selected from the group Chloroquine, Amodiaquine, mepacrine and Sontaquine.
 10. Use according to one of the claims 1 to 5, characterised in that antifolates are used as active substances for resistance-free therapy of malaria or other infections caused by Plasmodia.
 11. Use according to claim 10, characterised in that the antifolates are selected from the group pyrimethamine, chloroguanide, sulphones and sulphonamides.
 12. Use according to one of the claims 1 to 5, characterised in that chemotherapeutics, antibiotics and/or anti-infectives are used as active substances for resistance-free therapy of Leishmaniases.
 13. Use according to claim 12, characterised in that methotrexate is used as chemotherapeutic.
 14. Use according to one of the claims 1 to 13, characterised in that the 5-substituted nucleosides or the prodrugs thereof are used in concentrations such that a blood concentration of 0,01 to 10 μg/Ml results.
 15. Use according to claim 14, characterised in that the 5-substituted nucleosides or the prodrugs thereof are used in concentrations such that a blood concentration of 0.05 to 5 μg/ml results.
 16. Use according to one of the claims 1 to 15, characterised in that the active substances are used in concentrations which are standard for the infectious diseases.
 17. Use according to one of the claims 1 to 16, characterised in that the administration is effected by injection, orally, rectally, intravaginally, intranasally and/or by local application.
 18. Use according to one of the claims 1 to 17, characterised in that further additives are selected from the group of aqueous and non-aqueous solvents, stabilisers, suspension agents, dispersion agents and wetting agents.
 19. Use according to one of the claims 1 to 18, characterised in that further additives are selected from the group polyethylene glycoles, colourants and perfume agents.
 20. Use according to one of the claims 1 to 19, in the form of a fine powder, powder, suspension, solution, emulsion, salve or paste. 